Numerous methods have been employed for the determination of cholesterol in biological fluids, in which a reagent combines with cholesterol in the fluid to form a colored reaction product. The concentration of cholesterol in the sample is then determined by measuring the depth or intensity of the color.
Previously known colorimetric methods for determining cholesterol have suffered from a lack of sensivity, from interference such as may be caused by hemoglobin, bilirubin, and icteric and turbid specimens, from the instability or danger of the reagents utilized, or from the length of time required for the determination. The generally recoginized reference method of Abel et al, J. Biol. Chem. 195, 357 (1952) requires difficult and time-consuming saponification and ether extraction steps and is subject to variances caused by loss of cholesterol in the isolation process. The procedure of Jung et al, Clinical Chemistry 21, 1526 (1975) requires a dangerous reagent, uranyl acetate. A number of procedures have been based on the ferric chloride/sulfuric acid method of Zak, Am. J. Clin. Pathol. 27, 583 (1957). When sulfuric acid, acetic acid, and ferric ion are added to a sample of biological fluid, the ferric ion combines with cholesterol to form a characteristic color. Other components of the fluid, such as bilirubin, react with the reagent, however, in the well-known Hopkins-Cole reaction, to form interfering colors. Therefore, protein is first precipitated from the biological sample by the addition of ferric chloride and acetic acid. These procedures lack sensitivity and suffer from the instability of the reagents.